The mammary gland is a complex organ whose growth and development are controlled by the interaction of a wide variety of hormones and growth factors also involved in the etiology and progression of the cancerous state. Our emphasis has been on the interactions of prolactin (PRL), estrogen (E), and progesterone (P) during the peripubertal period and the lobulo-alveolar development of pregnancy as well as during tumorigenesis. We have shown that E and P are required to promote development of the primary/secondary ductal network in addition to other endocrine growth factor(s), and that P facilitates the formation of tertiary side-branches. The Hox-related homeobox containing gene, Msx2, is highly expressed during branching morphogenesis where our studies in vivo and in vitro showed that its expression is regulated by P in the presence of E. Concurrent with these morphological changes, progesterone receptor (PR) localizes at early branch points. During peripubertal morphogenesis PR distribution shifted from a homogeneous to a heterogeneous pattern.The transcription factor, C/EBP-beta appears to regulate mammary epithelial cell fate resulting in the correct spatial pattern of gene expression required to permit steroid hormone regulated cell proliferation.We demonstrated differential transcription of the four PRL receptor (PRLR) isoforms by stromal as well as epithelial cells throughout development. The distribution of the PRLR in the epithelium, like that of the PR, progressed from a homogeneous to a heterogeneous pattern. Hence, while exogenous P or PRL alone was without effect on epithelial proliferation in ovariectomized mice, these hormones synergize to stimulate epithelial and stromal proliferation. We have also identified that insulin-like growth factor-II (IGF-II) may mediate PRL-induced proliferation in the mammary gland. Expression of IGF-II is increased in ductal and alveolar epithelium during puberty- and pregnancy-associated morphogenesis, and IGF-II stimulates lobuloalveolar development in whole organ culture.We are studying changes in the vascular network that facilitates lactogenesis and tumorigenesis in the mammary gland. Our data support the conclusion that specific cell types within the mammary gland differentially transcribe VEGF and that it functions as an autocrine/paracrine endothelial growth factor under hormonal regulation. Additional studies aim to understand the role of PRL in the etiology and progression of human breast cancer. Specifically, we are examining the role of PRLR isoforms and autocrine/paracrine PRL in tumorigenesis and carcinogenic susceptibility. Comparisons between cancerous and adjacent, noninvolved tissue from the same breast of 23 patients showed that, on average, both PRL and PRLR mRNA expression was significantly higher in the cancerous tissue compared to the noninvolved tissue. The various forms of the PRLR differ in their cytoplasmic domains due to alternate splicing. We have cloned two new splice variants of the human prolactin receptor, PRLR-SFA (SFA) and PRLR-SFB (SFB). Both SFA and SFB were detected by RT-PCR in normal breast and breast tumor tissue. The PRLR-LF transduces the signal for both differentiation and mitogenesis. Neither SFA nor SFB is able to transduce a differentiation signal when stimulated with PRL. However, SFA was found to function as a strong dominant negative of the differentiation function of LF; SFB is a weak dominant negative.The role of the autocrine/paracrine system for prolactin in human breast cancer as well as the presence of the various receptor isoforms is under active investigation.